Our long-term goal is to elucidate the molecular mechanisms of regulated Cu efflux by two mammalian Cu-[unreadable] ATPases in polarized epithelial cells. ATP7A in intestinal epithelial cells delivers Cu to the circulation[unreadable] (basolateral environment) and ATP7B in hepatic cells delivers Cu to the bile (apical environment). Mutations[unreadable] in either protein cause human disease, emphasizing their importance in Cu-homeostasis. Based on[unreadable] extensive preliminary data in vivo and in vitro, we propose to test three hypotheses. In AIM 1 we[unreadable] hypothesize that in Cu-loaded hepatic and intestinal cells, Cu-ATPases pump Cu into unique vesicles that[unreadable] fuse with the basolateral (ATP7A) or apical (ATP7B) plasma membrane (PM) to release "stored" Cu. We will[unreadable] identify these compartments using immuno-EM and immuno-isolation/proteomic approaches. In AIM 2 we[unreadable] hypothesize that the chloride channel, CIC4, is an anion shunt for both Cu-ATPases in their secretory and[unreadable] efflux function/locations. In basal Cu, We will determine if Cu loading of ceruloplasmin in WIF-B cells by[unreadable] ATP7B and hephaestin in Caco-2 cells by ATP7A occurs in chloride-free conditions and after knock-down of[unreadable] CIC4. We will also examine the role of CIC4 in Cu efflux from the cells.In Aim 3, we hypothesize that when[unreadable] Cu levels are elevated, unique structural signals in the N-terminus of ATP7B regulate its trafficking/apical[unreadable] efflux function. Our test includes biochemically isolating and identifying new protein interactors, elucidating[unreadable] the role of a newly-identified modulator of Cu efflux (Murr1) on the dynamics/function of wt and selected N-terminal[unreadable] mutants of exogenous ATP7B, and using the LEG rat model of Wilson Disease to confirm and[unreadable] extend in vitro findings. Approaches will include RNAi and overexpression of wt modulators, as well as yeast[unreadable] two hybrid. Also in AIM 2 we hypothesize that when Cu levels are lowered, ATP7A and ATP7B are retrieved[unreadable] from their distinct compartments through recognition of structural signals in their C-termini. We will use yeast[unreadable] two hybrid and affinity isolation of putative protein interactors from extracts of relevant tissue.